1. A stochastic feedback loop model predicts the kinetics of DDR and growth arrest at the single cell level.
A stochastic feedback loop model predicts the kinetics of DDR and growth arrest at the single cell level. (A) Feedback loop model. Uncapped telomeres (red) or unrepaired double strand breaks (black) trigger a DDR activating TP53 and CDKN1A. High CDKN1A levels initiate signalling through GADD45, MAPK14 and TGFβ leading to mitochondrial dysfunction and increased production of ROS, which damage nuclear DNA, thus inducing more non‐telomeric DNA damage foci, stabilizing DDR and growth arrest leading to a stable senescent phenotype. (B) Stochastic simulations. IR at t=0, SB from t=6 days. Results are M±s.d., n=500. The vertical line indicates start of SB treatment (inhibition of MAPK14 activity to 60%, compare Supplementary Figure S11). For CDKN1A, a threshold value at 2 s.d. above basal is indicated (dashed line). Experimental data (DHR flow cytometry for ROS, γH2A.X immunofluorescence for foci frequencies, CDKN1A/TUBULIN and p53 S15/total TP53 western, M±s.e.m., n⩾3) are shown in red for comparison. (C) The same simulation as in Figure 3B but completely without feedback after CDKN1A. (D) Effect of SB treatment at 94 h after IR on DNA damage foci frequencies per cell as predicted by the stochastic model. M (black), s.d. (grey), n=500. (E) Confocal time series of an MRC5 cell expressing AcGFP–53BP1c at the indicated times (in h) after 20 Gy IR. SB was added at t=94 h (+SB). Images are compressed stacks (maximum intensity projections), focal depth=4.5 μm, with grey values converted to a colour scale as indicated in the lookup table. Full resolution time series is given in Supplementary Movie SM1. Green arrows indicate the one focus that is persistent over the whole observation period in this cell. All other foci are transient, and red arrows mark examples of these. (F) Frequencies of AcGFP–53BP1c foci after IR and under SB treatment as measured by live cell microscopy. M (black), s.e.m. (dark), s.d. (light), n=22. The vertical bar indicates the start of treatment with SB at t=94 h. (G) Kaplan–Mayer survival curves for AcGFP–53BP1c foci in young, senescent and irradiated MRC5 cells. Numbers of foci analysed are between 61 and 146 per treatment from two independent experiments. ***P=1.1 × 10−12 (Cox regression). (H) Frequencies of long‐ and short‐lived AcGFP–53BP1c foci per cell (all irradiated with 20 Gy) before (control) and after start of treatment with SB M±s.e.m., n=275–354. *P=0.006; NS, not significant (Students’ t‐test).
João F Passos et al. Mol Syst Biol 2010;6:347
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